PROJECT 3: PROJECT ABSTRACT/SUMMARY Urinary tract infections are the most common medical problem in Urology, and one of the most pervasive medical illnesses. Patient presentation falls along a spectrum of intensive, lower urinary tract symptoms, to transient less defined discomfort and non-diagnostic urinary findings. We believe the spectrum of presenting symptoms results from different sites of colonization of bacteria and different levels of virulence at those sites. Our preliminary data indicates the bacterial tropism and virulence is directly related to the capacity of bacteria to obtain nutrients, and the most coveted substance is iron. Iron is a ?precious metal? for bacteria because all metabolic processes, including energy production and cell division requires 100,000 atoms per bacteria. The urinary system is a particularly intriguing site of iron acquisition, because while the urine fluid contains only Nano-Molar iron content, the urine also contains 106 red blood cells/day, containing 109 heme iron atoms each. In this setting of iron starvation, yet potentially heme-iron abundance, bacteria can rapidly deploy tools to transfer iron from mammalian proteins, and from heme rings directly across their membrane. We propose that this setting is dominated by heme iron, and that heme transport systems take priority initially in stealing our iron. This hypothesis not only derives from analysis of bacterial gene expression, but also our study of the epithelial response to the invasion of the bladder. By creating novel tools to isolate snapshots of nascent RNA, we discovered that the urothelium and specialized cells elsewhere in the urinary system activate their own system of heme capture, heme metabolism, and iron sequestration. These activated mammalian pathways are of great interest because of the immediacy of their responses, the recruitment of novel heme transporters at the site of bacterial attack, and the production of the heme product, Carbon Monoxide, a bacteriostatic agent. In addition, these pathways not only decontaminate heme, but they are the core complex of the Circadian Clock. We are cognizant that the implications of these findings will require considerable research, but they include our finding that the apical membrane of the bladder, the urothelium, can transport heme with a novel mechanism and the realization that bacteria stimulate this process as a mechanism of innate immune defense known as nutritional immunity. As a result of these mechanisms, we found that night time and day time UTI generate different biological outcomes. In this renewal proposal for Project 3 of the Columbia University George M. O?Brien Urology Research Center we go back to basic iron biology and we carefully document heme and iron transport across the bladder, cell and bacterial responses using novel imaging tools and novel iron and CO capture tools, mouse ko?s and bacteria carrying mutations in iron pathways that are critical in mice and human infection. We work with leading microbiologists (Uhlemann), geneticists (Mendelsohn, Gharavi) and lead scientist in iron biology (Hamza). Together, our studies will demonstrate that the urothelium is a metabolically active cell layer that uses iron biology to detoxify the daily microscopic hematuria and for immune defense upon their invasion.